Effect of a Subunit on Allosteric Modulation of Ion Channel Function in Stably Expressed Human Recombinant g-Aminobutyric AcidA Receptors Determined Using Cl Ion Flux

Inhibitory g-aminobutyric acid (GABA)A receptors are subject to modulation at a variety of allosteric sites, with pharmacology dependent on receptor subunit combination. The influence of different a subunits in combination with b3g2s was examined in stably expressed human recombinant GABAA receptors by measuring Cl influx through the ion channel pore. Muscimol and GABA exhibited similar maximal efficacy at each receptor subtype, although muscimol was more potent, with responses blocked by picrotoxin and bicuculline. Receptors containing the a3 subunit exhibited slightly lower potency. The comparative pharmacology of a range of benzodiazepine site ligands was examined, revealing a range of intrinsic efficacies at different receptor subtypes. Of the diazepam-sensitive GABAA receptors (a1, a2, a3, a5), a5 showed the most divergence, being discriminated by zolpidem in terms of very low affinity, and CL218,872 and CGS9895 with different efficacies. Benzodiazepine potentiation at a3b3g2s with nonselective agonist chlordiazepoxide was greater than at a1, a2, or a5 (P , 0.001). The presence of an a4 subunit conferred a unique pharmacological profile. The partial agonist bretazenil was the most efficacious benzodiazepine, despite lower a4 affinity, and FG8205 displayed similar efficacy. Most striking were the lack of affinity/ efficacy for classical benzodiazepines and the relatively high efficacy of Ro15-1788 (53 6 12%), CGS8216 (56 6 6%), CGS9895 (65 6 6%), and the weak partial inverse agonist Ro15-4513 (87 6 5%). Each receptor subtype was modulated by pentobarbital, loreclezole, and 5a-pregnan-3a-ol-20-one, but the type of a subunit influenced the level of potentiation. The maximal pentobarbital response was significantly greater at a4b3g2s (226 6 10% increase in the EC20 response to GABA) than any other modulator. The rank order of potentiation for pregnanolone was a5 . a2 . a3 5 a4 . a1, for loreclezole a1 5 a2 5 a3 . a5 . a4, and for pentobarbital a4 5 a5 5 a2 . a1 5 a3. The inhibitory neurotransmitter GABA plays a major role throughout mammalian central nervous system via activation of GABAA and GABAB receptors. GABAA receptors are multisubunit oligomers with an integral chloride channel belonging to the ligand-gated ion channel receptor family. To date, cloning studies have revealed the gene family encompasses a multitude of different subunits, falling into seven families (a1–6, b1–3, g1–3, d, e, p, and u) (Whiting et al., 1999). Studies on subunit assembly and stoichiometry suggest the subunits coassemble as pentamers to form a variety of receptor subtypes that are differentially expressed in mammalian brain (Wisden et al., 1992; Fritschy and Mohler, 1995; McKernan and Whiting, 1996). In vitro, an a, a b, and a g subunit are required to form fully functional GABAA receptors resembling those in native tissues (Pritchett et al., 1989) where 2:2:1 stoichiometry is often observed (Im et al., 1995; Chang et al., 1996), although (a)2(b)1(g)2 may also be possible (Sieghart et al., 1999). The presence of different subunits confers different pharmacological, functional, and modulatory properties on receptor subtypes. The largest population of GABAA receptors in rat brain has subunit composition of a1b2g2, whereas a2b3g2 and a3bg2/g3 together constitute the next most prevalent subtypes (McKernan and Whiting, 1996). There is also a variety of other combinations that form more minor receptor populations (Sieghart et al., 1999), although the precise physiological roles of many of these are not fully defined. GABAA ion channel function can be modulated via a variety of allosteric sites (Sieghart, 1992) that are the target for a number of therapeutic agents, such as benzodiazepines, barbiturates, steroids, convulsants, and anesthetics (for review, see Sieghart, 1995; Rabow et al., 1995). Affinity and efficacy at the benzodiazepine site, one of the better characterized, are influenced by a and g (McKernan et al., 1995; Buhr et al., 1996; Wingrove et al., 1997) but not b subunits (Hadingham et al., 1993). The benzodiazepine site is the ABBREVIATIONS: GABA, g-aminobutyric acid; DMEM, Dulbecco’s modified Eagle’s medium; DIDS, 4,49-diisothiocyanatostilbene-2,29-disulfonic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid. 0026-895X/01/5905-1108–1118$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 5 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 340/899063 Mol Pharmacol 59:1108–1118, 2001 Printed in U.S.A. 1108 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from target for commonly prescribed anxiolytic drugs, occurring at the interface of an a(1–6) and a g(1–3) subunit, with residues in both influencing modulation (Smith and Olsen, 1995; Wingrove et al., 1997). The presence of g2 confers a classical benzodiazepine pharmacology to GABAA receptors (Pritchett et al., 1989), whereas receptors containing the less abundant g1 subunit (Benke et al., 1996) display atypical benzodiazepine pharmacology, including a much lower affinity for Ro15-1788 (Wafford et al., 1993). Introduction of a g3 subunit affects the affinity and efficacy of some but not all benzodiazepine ligands (Hadingham et al., 1995). Of the a subunits, the presence of a4 or a6 results in low affinity for classic benzodiazepines such as diazepam but high affinity for the imidazobenzodiazepine Ro15-4513 (Knoflach et al., 1996; Wafford et al., 1996). Cloning and stable expression of selected GABAA subunits permits detailed analysis of pharmacological profiles associated with specific receptor subtypes and this, together with the knowledge of which combinations predominate in vivo, their localization, and relative abundance (McKernan and Whiting, 1996), may help to dissect the physiological roles of these inhibitory receptors. We have examined the influence of subunit composition on activity at human recombinant GABAA receptors stably expressed in mammalian cells, with particular emphasis on compounds that modulate ion channel function. The methodology involves measuring Cl influx through the GABAA receptor ion channel pore and, in 96-well format, offers the potential for multiple manipulations simultaneously within the same experiment without the need for washout and recovery associated with electrophysiological experiments where a single cell or oocyte is patch-clamped. Many previous studies used primary cultures, synaptoneurosomes, or microsac preparations from native tissues to examine GABA-activated transmembrane ion flux (Kardos, 1993) but the precise array of subunits is generally unknown and the measurable response may arise from a mixed receptor population. The other primary techniques used to examine GABAA receptor function are electrophysiological, with much of the evaluation done in transient expression systems such as Xenopus laevis oocytes (Rabow et al., 1995). Comparisons of GABAA receptor function in stably transfected Ltk cells and transiently transfected oocytes reveal some differences, primarily with regard to the actions of ethanol (Harris et al., 1997). Stable expression of recombinant receptors, however, offers the advantage of a homogeneous clonal population of cells where expression can be carefully monitored, reducing the likelihood of misassembly and overcoming some of the variability seen with transient expression such as multiple transient assembly (Ebert et al., 1996), poor efficiency of transfection, and change of receptor expression with time. In this study, the pharmacological consequences of stably expressing different GABAA receptor a subunits in combination with b(3) and g(2s) in mouse Ltk cells are examined. Materials and Methods Tissue Culture and Cell Preparation. Human GABAA receptor subunits were stably expressed in mouse Ltk fibroblast cells by transfection of the appropriate subunit cDNAs in vector pMSGneo using standard calcium phosphate transfection techniques (Hadingham et al., 1992). Cells were maintained in stock trays in Dulbecco’s modified Eagle’s medium (DMEM) with 580 mg/l glutamine (Life Technologies Europe, Paisley, Scotland, UK), supplemented with 10% (v/v) FetalClone II serum (Hyclone, Logan, UT) and 1 mg/ml geneticin. Stocks were split 1:8 into fresh trays once a week. For seeding, cells (passage 5–25) were removed from stock plates with 0.05% trypsin/0.53 mM EDTA solution (Life Technologies, Gaithersburg, MD) and resuspended in DMEM supplemented with 10% FetalClone II serum but without geneticin. Cells with subunit composition anb3g2 were seeded into Costar 96-well plates at densities of between 3 and 8 3 10 cells/ml in a volume of 200 ml/well and grown in the presence of 10% serum for 5 to 8 days in an incubator at 37°C. Receptor expression, which is under the control of a dexamethasonesensitive promoter, was induced 24 h before experiment in the confluent cell monolayers using serum-containing DMEM supplemented with 1 mM dexamethasone (Sigma Chemical, Poole, Dorset, UK). Early Optimization of Flux Conditions. The assay was developed initially in cells grown on 25-mm coverslips contained in sixwell plates. Coverslips were immersed in HEPES/Krebs’ buffer containing Cl and appropriate drugs for 5 s, and then washed in ice-cold stop buffer (HEPES/Krebs’ buffer containing 100 mM picrotoxin, pH 7.4 at 4°C using 1 M Tris) and influx determined by digestion in NaOH and scintillation counting. Although agonist responses and benzodiazepine modulation could be measured, the signal was small and the methodology time-consuming. Assay conditions were optimized, including examining the effects of assay temperature, extracellular chloride concentration, optimizing agonist exposure times, and using chloride transport inhibitors. The assay was then transferred into 96-well format to improve throughput. Cells were initially seeded at densities of 2 3 10cells/ml in a volume of 200 ml/well with 7 days of induction. Following cell washing, ligand, GABA, and test compounds were added simultaneously for 10 s to each well in a 50-ml volume (n 5 6 wells/treatment), followed by aspiration and washing with stop buffer. Effects of inducing receptor expression with dexamethasone for different periods were investigated. A progressive improvement in the signal was observed as days of induction were reduced from 7 days to 1 day, with benzodiazepine modulation by flunitrazepam being optimal at 1 day. The current protocol was therefore adopted in which cells are seeded and allowed to grow for 5 to 8 days before being induced 1 day before use. The assay procedure was also amended at this stage to include a 30-s preincubation step with test compounds to allow equilibration before addition of GABA and Cl for a further 7 s. Measurement of Cl Influx. Following the 24-h induction period with dexamethasone, 96-well plates were removed from the incubator. Cells were initially washed at room temperature with buffer of the following ionic composition 162 mM NaCl, 6.4 mM KCl, 1.7 mM MgCl2, 1.2 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM HEPES, pH 7.4, using a Dynatech Ultrawash Plus 96-well plate washer. Induction medium was aspirated and, after a prewash, cells were washed twice for 2-min periods, aspirating after the final wash. Measurement of Cl influx was performed in assay buffer where chloride ions were absent to favor uptake of radioactive chloride. Chloride salts were replaced by acetate salts and the chloride-free assay buffer, pH 7.4, at 4°C using 1 M Tris) also contained 11 mM glucose and the chloride transport inhibitors DIDS (10 mM) and furosemide (100 mM). Modulatory compounds or assay buffer was added to the wells in a volume of 40 ml for a 30-s preincubation using a Robbins Hydra 96 dispenser. At t 5 30 s a subsequent 40-ml addition of a 23 (Cl ligand solution 6 GABA in chloride-free assay buffer) was then made to each well using a Sagian Beckman Multipette 96-well dispenser, incubating for a further 7 s. At t 5 37 s Cl influx was terminated by aspiration of incubation buffer and immediate washing with 53 200 ml of ice-cold picrotoxin stop buffer using a second Dynatech plate washer. Stock Cl ligand was supplied by Amersham Pharmacia Biotech (Buckinghamshire, UK) as NaCl in aqueous solution (122 mg Cl/ml) at a concentration of 1 mCi/ml. The final concentration of radioligand in the assay was 4.44 mCi/ml. Influence of a Subunit on GABAA Receptor Ion Flux 1109 at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from Scintillation fluid (200 ml) was finally added to each well, the plate sealed, and influx of Cl was determined by scintillation counting on a Packard TopCount. Data Handling and Analysis. Cl influx was measured on each plate in replicates of six wells per treatment. Each 96-well plate included the following controls: six wells for basal or background influx in the absence of GABA mimetics (chloride-free buffer plus Cl only); six wells treated with a maximal concentration of GABA (buffer plus Cl solution containing GABA, 100 mM final concentration); and for modulation of GABA-stimulated flux, six wells treated with Cl plus an EC20 concentration of GABA. For benzodiazepine modulation, each plate also contained a full benzodiazepine agonist in the presence of an EC20 concentration of GABA as an internal standard, relative to which everything else was expressed, to control for any minor variations in cell responsiveness from day to day. For a1, 2, 3, and 5, this was the nonselective benzodiazepine agonist chlordiazepoxide (30 mM). Modulation at other sites on the GABAA receptor is shown as the amount of potentiation of the EC20. Agonist stimulation and modulatory responses were assessed by performing one-way analysis of variance using Prism version 2.0b (GraphPad Software, San Diego, CA) followed by post hoc testing using the Bonferroni test to determine significance. Concentrationresponse curves were fitted by nonlinear least-squares regression analysis using RS1 (BBN Research Systems, Cambridge, MA). Mean EC50, IC50, and efficacy values are presented as the arithmetic mean and standard error of the mean from a number of independent determinations. Data from experiments to determine the relative efficacy of modulatory agents were calculated using ActivityBase, Fig. 1. Optimization of flux assay using a1b3g2 cells. a–c, performed in coverslip format with stimulation by 10 mM muscimol. a, effect of varying [Cl2]ext. b, time course at 4°C in the presence of 8 mM [Cl 2]ext. c, effect of DIDS and furosemide on 4°C time course. d, effects of DIDS and furosemide on signal in final 96-well format [3 3 10cells/ml, induced 1 day, [Cl2]ext 5 0, 4°C, 30-s preincubation, 10-s ( Cl 6 GABA) addition]. 1110 Smith et al. at A PE T Jornals on O cber 3, 2017 m oharm .aspeurnals.org D ow nladed from version 3.2.0 (ID Business Solutions Ltd., Guildford, Surrey, UK) and Microsoft Excel, version 5.0 (Microsoft, Redmond, WA). Benzodiazepine binding affinities quoted represent displacement of specific binding of [H]Ro15-1788 for receptors containing a1, 2, 3, and 5 subunits and displacement of [H]Ro15-4513 binding for a4b3g2 receptors. Whole-Cell Patch-Clamp of Ltk Cells. Responses to GABA were also determined electrophysiologically in Ltk cells stably expressing a1-5b3g2 GABAA receptors after 24-h induction with dexamethasone. Glass coverslips containing a cell monolayer were placed in a chamber on the stage of a Nikon Diaphot inverted microscope and perfused continuously with artificial cerebral spinal fluid containing 149 mM NaCl, 3.25 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 11 mM D-glucose, D-(1)-sucrose, pH 7.4, and observed with phase-contrast optics. Fire-polished patch pipettes were pulled using conventional 120TF-10 electrode glass and tip diameter was approximately 1.5 to 2.5 mM, with resistances around 4 MV. The intracellular solution contained 130 mM CsCl, 10 mM HEPES, 10 mM BAPTA.Cs, 5 mM ATP.Mg, 0.1 mM leupeptin, 1 mM MgCl2, 100 mM NaVO3, pH adjusted to 7.3 with CsOH and 320 to 340 mOsm. Cells were voltageclamped at 260 mV via an Axon 200B amplifier (Axon Instruments., Foster City, CA) and drugs applied via a multibarrel drug delivery system, which could pivot the barrels into place using a stepping motor, ensuring rapid application and washout of drug. GABA was applied to the cell for 5 s with a 30-s washout period between applications. Noncumulative concentration-response curves to GABA were constructed and curves fitted using a nonlinear least-squares-fitting program to the equation f(x) 5 Bmax/[1 5 (EC50/x) H], where x is the drug concentration, EC50 is the concentration of drug eliciting a half-maximal response, and nH is the Hill coefficient.